Team:CIEI-BJ/Proof
Our Idea
What's our idea?
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Our idea
SmCPS1 is the first and one of the most important enzymes in Tanshinone synthesis. It converts geranyl geranyl pyrophosphate(GGPP) into copalyl diphosphate (CPP).
To produce the enzyme SmCPS1 through synthetic biology, we constructed the gene circuit above. tac promotor, a strong hybrid promotor, is used to control and increase the expression levels of a target gene and is used in the over-expression of recombinant proteins, produced from the combination of promotors from the trp and lac operons.
Thrombin protease and TEV are the two type of restriction enzyme cleavage sites which can let SmCPS1 gene insert in between and later help the SmCPS1 enzyme to be purified. GFP was used to test the expression of our target gene SmCPS1. The target gene (SmCPS1) was obtained from Institute of Botany, Chinese academy of Science.
In our research, we used pGEX-KG as the vector of our Biobrick device. It basically composes of Ampicillin anti-fragment, lac promoter with different primers, and tac promoter followed by multiple cloning site, which offers a favorable system to further insert our target DNA (SmCPS1) via pre-existed thrombin protease and newly added TEV.
To avoid our target gene be digested by the restriction enzyme after we combine it with the plasmid iGEM provide, we did site-directed mutagenesis
Our method
